DESCRIPTION: Adeno-associated viruses (AAV) have demonstrated great promise for gene therapy of a variety of heart diseases. Several AAV variants, such as AAV serotype 9 (AAV9), have been identified with high delivery efficiencies in cardiac cells. Unfortunately, the vectors still suffer from significant delivery to non-target sites, such as liver and skeletal muscles. Consequently, AAV vectors may need to be delivered via invasive administration routes to physically localize gene delivery to the heart. Most current strategies to achieve targeted delivery focus on enabling vectors to bind cell surface receptors that are overexpressed on target diseased cells. Unfortunately, identification of single receptors that lead to high delivery specificity has been difficult to achieve. As an alternate strategy, we hypothesize AAV vectors can be designed to target extracellular proteases - a hallmark of several cardiovascular diseases, including myocardial infarction. The protease-activated viruses (PAVs) will be unable to transduce cardiac cells until they detect pathological concentrations of specific extracellular proteases. To enhance the gene delivery specificity, we will combine transcriptional control with protease activation. In particular, the PAVs will carry a transgene driven by a heart- specific promoter. If only transcriptional control is used, the gene expression will occur in all heart tissues. If only protease-activation is used, then gene expression may occur in off-target sites also exhibiting elevated protease levels. By combining transcriptional control with protease-activation, we aim to limit gene expression to heart tissues that are also diseased. We have promising proof-of-concept data both in vitro and in vivo showing we have successfully created the first generation of PAVs. At low levels of proteases, the vectors are unable to bind their cell surface receptor and display negligible cellular transduction. Upon exposure to extracellular proteases, either a single target protease or a combination of two different proteases, the PAVs switch on their cell receptor binding behavior and dramatically increase their transduction efficiency. In aim 1, we will generate a toolkit of single- or dual-input AAV9-based PAVs that are responsive to extracellular proteases known to be elevated in myocardial infarction. In aim 2, we will conduct molecular imaging with protease-activatable contrast agents to detect protease activity in the infarcted heart. The imaging studies will help guide and validate the design and performance of protease-targeted AAV9 vectors. If successful, the studies proposed here will lay the preclinical groundwork for the development of vectors that can deliver genetic therapies specifically to diseased heart tissues.